Pain and Analgesia (A*)

Question 1

Give two examples of electrode therapies for pain.

Where are the electrodes implanted in each case?

Answer 1

• For phantom limb pain, electrodes are inserted in the:

1 - Sensory thalamus.

and

2 - Periaqueductal grey area (remember the PAG is the primary control center for descending pain modulation).

• For trigeminal neuropathy, electrodes are implanted over the motor cortex corresponding to the painful area (in this case, the face).

Question 2

Define pain.

Answer 2

Pain is a combination of nociceptive and affective (emotional) components.

*Important because it implies that the nociception needs to be processed and the individual must be aware of the sensation for it to be considered ‘pain’. I.e. ‘pain’ and nociception are different.

Question 3

What are ‘innocuous’ and ‘noxious’ pain stimuli?

Answer 3

• ‘Innocuous’ stimuli cause weak intensity nociceptive firing, and therefore do not result in any pain sensation (they still stimulate the nociceptive fibres, however only lightly).
• As the nociceptive stimulus intensity increases, a threshold is reached where pain sensation begins. The stimulus is ‘noxious’ if it produces pain (passes the ‘threshold’).
• I.e. both innocuous and noxious stimuli stimulate nociceptive fibres, but whilst innocuous stimuli do not produce pain, noxious stimuli do.

Question 4

What is allodynia?

What is hyperalgesia?

Give an example of a cause of allodynia and hyperalgesia.

Answer 4

• Allodynia is the reduction in pain threshold for noxious stimuli, causing a previously innocuous stimulus to be perceived as a noxious one.
• Hyperalgesia is the increase in / hypersensitisation of pain sensation from a stimulus due to the decrease in pain threshold for noxious stimuli (previously painful stimuli are now even more painful).
• Allodynia and hyperalgesia occur in injury.

Question 5

How does prolonged physiological pain differ from chronic pain?

A*: What is the epidemiology of chronic pain?

Answer 5

• Prolonged physiological pain is hyperalgesia / allodynia maintained by afferent input, and is resolved by wound healing.
• Chronic pain is pathological hyperalgesia / allodynia that continues beyond the normal healing time and without afferent input. It is caused by central neuroplastic changes.
• Chronic pain is present in approximately 35% of all individuals over the age of 18 in the UK.

Question 6

What is wind-up?

Answer 6

• Wind-up is the increase in excitability of neurones in the dorsal horn of the spinal cord in response to repetitive input.
• It isn’t used much today because it was just found to be a form of long-term potentiation.

Question 7

List 6 causes of chronic pain.

Answer 7

Chronic pain can be caused by:

1 - Peripheral nerve injury.

2 - Diabetic neuropathy.

3 - Postherpetic neuralgia (caused by Shingles, the virus can re-emerge later in life, especially if the patient is immunocompromised).

4 - Multiple sclerosis.

5 - AIDS.

6 - Stroke affecting the thalamus.

Question 8

Define neuralgia.

Answer 8

Neuralgia is a chronic pain condition characterized by recurrent brief episodes of pain affecting the sensory region of a particular nerve (e.g. trigeminal neuralgia affects the face).

Question 9

List the types of primary sensory neurones.

What is the function of each neurone?

Answer 9

Types of primary sensory neurones include:

1 - A beta fibres (touch).

2 - A delta fibres (mechanical and thermal nociception).

3 - C fibres (polymodal nociception: mechanical, thermal and biochemical).

Question 10

Give an example of a common pain signal that stimulates primary sensory neurones in nociceptive pathways.

Answer 10

ATP is a common pain signalling molecule that stimulates primary sensory neurones in nociceptive pathways.

Question 11

How might spinal cord reorganisation cause allodynia?

Answer 11

• Abeta fibres can form axon sprouts that make connections in lamina II.
• This means the nociceptive pathway is able to receive input from low-threshold mechanoreceptors, a form of allodynia.

Question 12

What is neurogenic inflammation?

How might neurogenic inflammation cause peripheral and central sensitisation?

Answer 12

• Neurogenic inflammation is the process by which mediators are released directly from the cutaneous nerves to initiate an inflammatory reaction and sensitise nociceptive signalling.
• Mediators include CGRP, substance P, ATP and fractalkine.
• This results in the formation of an ‘inflammatory soup’, containing histamine, 5-HT H+, prostaglandins, TNF-alpha, bradykinin and cytokines can sensitise the ascending pain pathway:

1 - The substances in the inflammatory soup can cause sensitisation by phosphorylating transducer channels, including Nav1.8, and voltage-independent channels such as TRPV1 channels.

• These are the channels that normally cause depolarisation in the sensory neurones. Phosphorylation lowers the threshold for opening, causing peripheral sensitisation.

2 - The substances in the inflammatory soup can activate gliotransmitter receptors, resulting in cytokine release from glia.

• Cytokines such as IL-1 beta, IL-6 and TNF-alpha drive neuroplastic changes that cause central sensitisation (A*: through mechanisms such as heterosynaptic facilitation).

Question 13

What evidence exists for allodynia being caused by the loss of inhibitory gating interneurones?

Give an example of a mechanism to explain how inhibitory gating interneurones might be lost, and how this might result in allodynia.

Answer 13

• If bicuculline, a GABA antagonist, and strychnine, a glycine antagonist, are administered intrathecally into healthy individuals, allodynia occurs.
• This is because the inhibitory interneurones gating the spinal cord are blocked, allowing low nociceptive thresholds to produce pain.
• One suggested mechanism by which the inhibitory gating interneurones might be lost is the loss of the K+/Cl- exporter 2 pump.
• This is the pump necessary for maintaining high extracellular Cl-, so that GABA receptors can cause Cl- influx, causing inhibition (see inhibitory amino acids lecture).
• If this is lost, Cl- efflux occurs, causing excitation. The inhibitory action of the interneurones is now flipped and spinal gating interneurones now potentiate pain rather than inhibiting it.

Question 14

What is a neuroma?

Describe the process of neuroma formation.

Describe the changes in expression in a neurone with a neuroma.

Why do neuromas cause pain?

Answer 14

• A neuroma is a benign growth of nervous tissue.
• When an axon is damaged, axon sprouts grow from the neurone to repair the site of injury.
• If the site of injury is not reached by the axon sprouts, the axon sprouts persist and the nerve becomes surrounded by a lump of nerve endings formed from the axon sprouts.
• All of the proteins synthesised at the soma destined for the synapse therefore accumulate in the neuroma. There are also changes in expression:
• Galanin, VIP and neuropeptide Y increase in expression.
• Substance P and CGRP decrease in expression.
• Expression of Nav1.3 TTX-resistant sodium channels increases (see A* card 36 for reason), and so they tend to cluster at the neuroma. Nav1.8 and Nav1.9 channels tend to decrease in expression.
• Neuromas cause pain because the Nav1.3 TTX-resistant sodium channels ectopically discharge, causing action potential generation.

Question 15

Summarise the the pathophysiology of chronic pain by listing 3 peripheral and 4 central mechanisms.

Answer 15

Peripheral mechanisms for chronic pain:

1 - Sensitisation of nociceptors.

2 - Silent nociceptor activation (sensory afferents that do not respond to nociceptive stimuli unless injury or some other pathological process has occurred).

3 - Ectopic activity at the site of injury (e.g. with neuromas).

Central mechanisms for chronic pain:

1 - Sensitisation of spinal neurones.

2 - Loss of spinal inhibition.

3 - Neuroplastic changes altering the network activity of neurones in ascending pain pathways (e.g. mechanoreceptors synapsing on pain pathways).

4 - Serotonergic descending facilitation.

Question 16

List 5 possible pharmacological approaches to achieve analgesia.

Answer 16

Pharmacological approaches to achieve analgesia include:

1 - Targeting the opioid system (intrinsic analgesia system).

2 - Anticonvulsant drugs used as adjuvant analgesics.

3 - Antidepressant drugs.

4 - Inhibiting neurotrophic factors.

5 - NSAIDs.

Question 17

Which neurotransmitters are used in the dorsolateral fasciculus to cause inhibition of the ascending pain pathways in the dorsal horn?

Answer 17

The neurones of the dorsolateral fasciculus use 5-HT and enkephalin (an endogenous opioid) to cause inhibition of the ascending pain pathways in the dorsal horn.

Question 18

List 7 problems with using opioids for analgesia.

Answer 18

Problems with opioids for analgesia:

1 - Opioids decrease GIT motility, causing constipation.

2 - Opioids depress respiratory centres, posing a risk for overdose.

3 - Tolerance can develop for opioids, meaning doses must be escalated.

4 - Patients can develop dependence for opioids.

5 - Opioid-induced hyperalgesia can be caused by opioid-induced glial activation.

6 - Neuropathic pain is inherently resistant to opioid analgesia. Intolerably high doses must be given to achieve analgesia.

7 - Some opioids show biased agonism, the phenomenon whereby a ligand preferentially activates one of several signaling pathways, whereas another agonist in the same system on the same receptor preferentially activates another pathway.

Question 19

Why do some anticonvulsant drugs have analgesic properties (and hence can be used as adjuvant analgesics)?

List 5 drugs with these properties.

Answer 19

• Some anticonvulsant drugs have analgesic properties because they prevent high frequency firing in damaged neurones that turn out to also form part of the ascending pain pathways. Examples include:

1 - Carbamazepine.

2 - Lamotrigine.

3 - Gabapentin.

4 - Pregabalin.

5 - Ziconotide.

Question 20

Describe the mechanism of action of gabapentin and ziconotide as antiepileptics and analgesics.

Answer 20

• Gabapentin is an antiepileptic drug that binds to the alpha 2 delta subunit of voltage-gated Ca2+ channels in pathways which exhibit high frequency firing in epilepsy.
• These neurones also turn out to form part of the ascending pain pathways.
• Ziconotide is an analgesic drug with a similar mechanism. It is a selective N-type voltage-gated Ca2+ channel (AKA Cav2.2) blocker. These channels are involved in transmission in many pathways, including pain.
• Gabapentin has nothing to do with GABA (but they used to think it did).

Question 21

List 3 antidepressants that cause analgesia.

Give an example of a problem with these drugs as analgesics.

Describe the mechanism for this analgesia.

Answer 21

Antidepressants causing analgesia include:

1 - Amitriptyline.

2 - Venlafaxine.

3 - Bupropion.

• A problem is that amitriptyline has many side effects. Next generation antidepressants (#2 and #3) might be more useful in this regard.
• The exact mechanism is unknown, but is thought to involve enhancement of descending pain inhibition.
• The effects of noradrenergic antidepressants are thought to be more beneficial, as evidenced by the fact that TCA antidepressants are more effective than SSRI antidepressants.

Question 22

List 6 novel analgesic drug classes.

Answer 22

Novel analgesic drug classes:

1 - Selective Na+ channel blockers.

2 - TRPV1 antagonists.

3 - Cannabinoids.

4 - Inhibitors of neurotrophic factors (e.g. NGF and BDNF - see A* cards).

5 - Glial modulators.

6 - Cytokine/chemokine receptor antagonists.

Question 23

A*: What are MOR-biased ligands?

Give an example of a MOR-biased ligand.

Give an example of a challenge associated with measuring biased agonism.

Answer 23

• Mu-opioid receptor (MOR)-biased ligands are a safer form of opioid analgesic drugs.
• Opioids are commonly associated with respiratory depression.
• This is due to the effect of opioid receptors on respiratory centres of the brain. Specifically, studies in mice show that knockout of beta arrestin 2, a scaffold protein that regulates GPCR signalling, attenuates morphine-induced respiratory depression.
• MOR-biased ligands are able to activate MORs without triggering the downstream cascades that involve beta arrestin 2.
• Analgesia can therefore be achieved without causing respiratory depression, which is a particular risk with opioid overdose.
• An example of a MOR-biased ligand is oliceridine.
• A challenge associated with measuring biased agonism is the fact that the measured response for a particular cellular pathway depends on the assay being used. Therefore, when comparing results from multiple different assays, there is a need to normalise the results before being able to calculate a bias factor.
• This normally involves repeating the assay with an agonist that possesses maximum efficacy, and then comparing the response with the ligand of interest to calculate the % of max response for the ligand of interest. This value can then be compared across assays. This is known as the operational model, which was first demonstrated by Black and Leff (1983).

Question 24

A*:

Describe a mechanism that is thought to underlie the paradoxical allodynic and hyperalgesic effect of morphine.

Answer 24

Mechanism to explain the paradoxical hyperalgesic effect of morphine:

• Morphine binds to mu receptors in lamina I sensory neurones.
• Activation of mu receptors upregulates P2X4 receptor expression in these neurones.
• Activation of P2X4 receptors results in BDNF release.
• BDNF downregulates KCC2 transporter expression.
• This disturbs Cl- homeostasis, and therefore interferes with the functioning of inhibitory neurotransmitters (remember you start to get Cl- efflux rather than influx).
• The inhibitory gating GABA and glycine neurones lose their tonic inhibitory gating effect, causing allodynia and hyperalgesia.

Question 25

A*:

Describe the course and distribution of primary afferent neurones in the dorsal horn of the spinal cord.

Answer 25

• Primary afferent neurones enter the white matter of the dorsal horn via the dorsal roots.
• Once in the dorsal horn, axons carrying pain and temperature information form the dorsolateral tract of Lissauer, which comprises both ascending and descending collaterals.
• These collaterals travel 1-2 spinal levels (up or down) and enter the grey matter of the dorsal horn.
• In the grey matter, the collaterals synapse with second-order neurones in Rexed laminae I and II (the two most ventral grey matter regions):

1 - Rexed lamina I is the marginal zone.

2 - Rexed lamina II is the substantia gelatinosa.

• Information carried by second-order neurones in the substantia gelatinosa is carried further to Rexed laminae IV, V and VI, known collectively as the nucleus proprius. The nucleus proprius also receives additional innervation from the first-order neurones directly.
• Second-order neurones from Rexed laminae I-VI (marginal zone, substantia gelatinosa and the nucleus proprius) ascend the spinal cord to the thalamus as the spinothalamic tract (AKA anterolateral system).

Question 26

A*:

List 2 mechanisms by which nerve growth factor (NGF) contributes to chronic pain.

Give an example of a mechanism by which NGF decreases pain transmission.

Give an example of a potential analgesic drug which targets NGF.

Answer 26

Mechanisms by which NGF contributes to chronic pain:

• NGF is signalling is elevated in chronic pain (and also following injury). This contributes to chronic pain because:

1 - NGF upregulates genes that code for proteins that potentiate the pain response, such as:

• Substance P.
• TRPV1.
• Nav1.8 and 1.9 Na+ channels.
• NGF was found to be upregulated in the DRG and PAG in rat models of pain (Violi et al., 2010).

2 - NGF promotes axon sprouting.

• Immediately following axotomy, NGF is unable to reach the target tissue, and therefore declines.
• In response to this decline, pain-activated glia, namely satellite cells, release an overcompensation of NGF.
• This promotes induction of sprouting following axotomy, leading to neuroma formation and spinal cord reorganisation.
• A potential analgesic drug targeting NGF is acetyl-L-carnitine (ALCAR), which was shown to reduce NGF to physiological levels in the Violi et al. paper.
• Anti-NGF antibodies are also useful in the animal models of pain which are insensitive to the analgesic effects of opioids and NSAIDs (da Silva et al., 2019). NGF-targeting drugs might be a potentially useful drug in treatment-resistant chronic pain, however the ubiquity of NGF in the CNS and PNS means that there will likely be many adverse effects of anti-NGF treatment.
• However, it has also been shown that NGF can decrease pain transmission by increasing expression of BDNF in the DRG, which in turn facilitates GABA release. Hence, it is probably not as simple as ‘NGF increases pain’.

Question 27

A*:

Describe the mechanism that underlies the analgesic effect of NSAIDs.

Answer 27

• NSAIDs inhibit COX enzymes, resulting in a decrease in prostaglandin expression.
• Prostaglandins are involved in the induction of inflammatory responses.
• By reducing inflammation, fewer inflammatory substances (e.g. histamine, H+, prostaglandins, TNF-alpha, bradykinin and cytokines) are available to sensitise nociceptors.
• See card 12.

Question 28

A*:

Give a mechanism, other than phosphorylation of transducer channels, by which bradykinin causes pain.

Answer 28

• Bradykinin can cause pain by activating phospholipase A2, which produces arachidonic acid from membrane lipids.
• This results in an increase in production of prostaglandins, promoting inflammation (a positive feedback loop since bradykinin is a product of inflammation).

Question 29

A*:

Give an example of how acidosis can be transduced into a pain signal.

How can this transduction mechanism be targeted to achieve analgesia?

Answer 29

• Acid-sensing ion channel 3 (ASIC3) is a proton-gated ion channel involved in chemosensation and mechanosensation.
• Acidosis can be induced by tissue injury and inflammation.
• Activation of ASICs in response to acidosis causes an inward current of Na+, Ca2+ and K+, generating excitatory postsynaptic potentials in peripheral nociceptors.
• ASIC knockout mice have been shown to have a reduced response to tissue acidosis-induced hyperalgesia (Deval et al., 2008).
• ASIC inhibitors have potential for use in pain induced by tissue injury (not neuropathic pain), e.g. in inflammatory conditions (e.g. infection, rheumatic disorders, lupus and MS).
• Black mamba venom was found to contain ASIC blockers known as mambalgins, which have the potential to be used as potent analgesics (Diochot et al., 2012).
• See A8 card 35 for TRPV1.

Question 30

A*:

Describe the role of cannabinoid receptors in pain signalling.

Answer 30

Background:

• CB receptors involved in the modulation of pain are found mostly on Adelta fibres, but also on some C fibres.
• Noxious stimuli increase endocannabinoid release.
• CB1 receptors are located both centrally and peripherally in immune cells.
• CB2 receptors are located peripherally in immune cells.
• Both CB receptors are Gi/o GPCRs.

Analgesia mechanism:

• CB1 receptors can attenuate synaptic transmission of ascending pain pathways (through the downstream effects of Gi/o).
• Both CB1 and CB2 receptors are expressed in various inflammatory cells, and have an analgesic effect in inflammatory hyperalgesia.
• This effect is through attenuating NGF-induced degranulation of mast cells, reducing inflammation-induced peripheral sensitisation.
• Therapeutically, CB1 agonists are not preferable because of the side effects associated with the modulation of central synaptic transmission.
• Selective CB2 agonists are a potential avenue for analgesic drugs.
• Tolerance and dependence are potential issues with these treatments.

Question 31

A*:

Describe the role of glia in chronic pain.

List 2 analgesic drugs that target glia.

Answer 31

• Glia do not directly mediate pain, but can enhance existing pain signals.
• Glia can be activated by a variety of neuronal factors, such as those released in the inflammatory soup (card 13) and paradoxically by opioids.

4 successive glial activation states have been reported in chronic pain (Ji et al., 2013):

1 - ‘Glial reaction’.

• Upregulation of mitogens.
• Hypertrophy.
• Proliferation.

2 - Activation of MAPK signalling, e.g. ERK, due to the activity of mitogens which are upregulated in the glial reaction.

• It has been shown that ERK signalling mediates central sensitisation in dorsal horn neurones, because ERK regulates glutamate and K+ transmission.

3 - Increased expression of ATP, intercellular connexin channels (AKA hemichannels) and chemokine receptors, and decreased expression of glutamate transporters.

• ATP influences pain signalling via P2Y receptors on neurones and glia.
• Hemichannels permit flow of small signalling molecules such as ATP, ions and microRNAs.
• Chemokines attract inflammatory cells, promoting an inflammatory response.
• Glutamate transporters are required for glutamate uptake from the synapse. This is an important process in the cessation of signal generation at glutamatergic synapses. Hence, reduced expression of glutamate transporters prolongs glutamate-induced pain signals.

4 - Increased expression of growth factors (e.g. NGF - card 26), chemokines, cytokines and other glial mediators.

• These changes (1-4) alter neurone-glial and glial-neurone communication, and hence contribute to chronic pain by causing both central and peripheral sensitisation.

Analgesic drugs that inhibit glial activation include:

1 - Propentofylline.

2 - Minocycline.

Question 32

A*:

Describe the mechanism of action of gabapentin as an analgesic drug.

Answer 32

• It is generally thought that the analgesic effect of gabapentin is due to inhibition of voltage-gated Ca2+ channels, preventing excitatory transmission in ascending pain pathways.
• This has been evidenced, for example, by the fact that gabapentin inhibits neurotransmitter release in neurones in vitro.
• Voltage-gated Ca2+ channels have 5 subunits: alpha 1, alpha 2, beta, gamma and delta. The alpha 2 and delta subunits are connected with a disulfide bond.
• The putative binding site of gabapentin is on the alpha 2-delta subunit of voltage-gated Ca2+ channels.
• There is also evidence for binding sites on Na+ leak channels and NMDARs.
• Gabapentin is an antiepileptic drug, so the mechanism of action reflects its ability to attenuate synchronous firing seen in epilepsy.

Question 33

A*:

Summarise the descending pain pathway.

What is descending serotonergic facilitation?

List the sites of action of opioids.

Answer 33

• When second-order neurones ascend the pain pathway, they make one synapse in the medulla and one in the thalamus:

1 - In the thalamus, the second-order neurones synapse with neurones that make connections with enkephalin-releasing neurones in the PAG.

2 - In the medulla, the second-order neurone synapses with enkephalin-releasing neurones in the nucleus reticularis paragigantocellularis (NRPG).

In both cases:

• The enkephalin-releasing neurones synapse with GABAergic neurones in the PAG, causing inhibition.
• The GABA neurones normally tonically inhibit glutamatergic neurones, so loss of the GABAergic inhibition means an increase in firing of the glutamatergic neurones.
• These glutamatergic neurones make excitatory descending connections with enkephalin-releasing neurones in the nucleus raphe magnus (NRM) in the medulla.
• These enkephalin-releasing neurones, like in the PAG and NRPG, inhibit GABAergic neurones.
• As before, the GABAergic neurones in the medulla normally tonically inhibit 5-HT neurones, so loss of the GABAergic inhibition means an increase in firing of the 5-HT neurones.
• These 5-HT neurones make excitatory descending connections through the dorsolateral fasciculus with enkephalin neurones in the dorsal horn, which in turn inhibit both the first-order and second-order ascending sensory neurones.
• Also, noradrenergic neurones from the locus coeruleus in the pons make descending connections with second-order sensory neurones, causing inhibition.
• Descending serotonergic facilitation is a form of central sensitisation present in chronic pain in which serotonergic neurones are pathologically hypoactive. This hypoactivity results in loss of inhibition of ascending pain signals by reducing activity of enkephalin-releasing neurones in the dorsal horn.

1 - Opioids inhibit nociceptor stimulation in the periphery.

2 - Opioids stimulate enkephalin-releasing neurones in the NRPG and PAG.

3 - Opioids inhibit enkephalin-releasing neurones in the dorsal horn.

Question 34

A*:

List 4 drugs that target the opioid system to achieve analgesia.

List 2 problems with opioid analgesics.

How can these problems be resolved?

Answer 34

Drugs targeting the opioid system to achieve analgesia:

1 - Morphine, an opioid receptor agonist.

2 - Codeine, an opioid receptor agonist.

3 - Nalorphine, a partial agonist for the mu opioid receptor.

4 - RB120, an enkephalinase inhibitor.

Problems with opioid analgesics:

1 - Opioids can cause dependence. This is due to the presence of opioid receptors in the VTA-NAc neurones, stimulating the mesolimbic reward pathway.

• Methadone is an opioid agonist at these receptors that can be used to treat opioid dependence. It is preferable because it does not produce euphoria, and it has a long-lasting action.

2 - Opioids cause respiratory depression. This is due to the presence of opioid receptors in the respiratory centres in the brainstem.

• One way of countering this clinically is with 5-HT4A agonists. 5-HT4A receptors have an opposing effect to opioid receptors in respiratory centres, but they do not oppose the analgesic effects of opioid receptors on the descending pain pathway.

Question 35

A*:

Briefly describe the structure of TRPV1 channels.

Discuss the role of TRPV1 channels in pain.

What is the potential for TRPV1-targeting drugs.

Answer 35

Structure of TRPV1:

• TRPV1 has 6 transmembrane domains.
• The channels generally arrange as either homomeric or heteromeric tetramers.
• The transmembrane segment between domains 5 and 6 of each subunit contributes to the ion pore.

Role of TRPV1 channels in pain:

Summary of points from cards above:

1 - Phosphorylation of TRPV1 in response to components of the inflammatory soup is part of the process of peripheral sensitisation in response to neurogenic inflammation.

2 - TRPV1 antagonists are a novel analgesic drug class.

3 (A*) - TRPV1 is upregulated by NGF, which is overexpressed in pain states.

New A* points:

• TRPV1 is involved in chemosensation and thermosensation.

1 - Chemosensation.

• TRPV1 responds to a number of chemical stimuli and toxins, for example capsaicin, allicin (a protein derived from garlic and onions) and spider toxins, and inflammatory stimuli, such as factors in the neurotrophic soup (see card 12 for a list).
• There is also evidence to suggest that TRPV1 responds to lactate, the substance that mediates acidosis following ischaemia. However, the role of TRPV1 in acidosis-induced pain is unclear:
• Evidence for the role of TRPV1 channels in the sensation of acidosis is abundant. For example, it has been demonstrated that lactate potentiates CGRP release from the spinal cord in mice (Wang and Fiscus, 1997), a pronociceptive signal. Since CGRP release is generally considered to be a TRPV1-dependent process, this finding would imply that lactate increases TRPV1 activity, resulting in increased nociception. Furthermore, acidotic conditions have been shown to cause a shift in the temperature-response relationship of TRPV1, resulting in sensitisation to inflammatory stimuli.
• However, more recent evidence by de la Roche et al. (2016) investigating the functions of the TRPV1 channel more directly found that lactate inhibits TRPV1. This study found that TRPV1 receptor potentials and TRPV1-induced Ca2+ influx are attenuated by addition of lactate.
• Hence, there is a need to contextualise the role of TRPV1 signalling in acidosis-induced pain, considering the interaction with other signalling cascades such as ASIC3 (see A* card 29), because although there is abundant evidence to suggest that TRPV1 channels are involved in acidosis, the mechanisms by which signal transduction occurs is unclear.

2 - Thermosensation.

• Thermosensation is a more putative function of TRP channels. It is mediated by a range of TRP channels. Specifically, TRPV1 channels are hot (>45 degrees celsius) polymodal nociceptors, TRPV3/4 receptors are hot (>45 degrees celsius) cutaneous thermoreceptors and TRPM8 channels are cold (<15 degrees celsius) cutaneous thermoreceptors.
• Thermosensation information is carried by C fibres.
• The C-terminus of the TRPV1 and TRPM8 channels (at the distal end of S6) is thought to be responsible for their heat-sensing properties. This is evidenced by a study by Brauchi et al., in which the C-termini of TRPV1 and TRPM8 channels were swapped, resulting in cold-sensing TRPV1 channels and heat-sensing TRPV1 channels.
• The ability of thermosensing TRP channels to detect changes in temperature lies in their increased susceptibility to changes in enthalpy compared to other sensory receptors. That is, they are able to more efficiently absorb thermal energy at a constant pressure. This thermal energy is converted into a conformational change, which in turn modifies the probability of depolarisation.

Potential TRPV1-targeting drugs:

• Counterintuitively, topical application of capsaicin-containing creams was shown to produce an analgesic effect in rheumatoid arthritis. This is likely due to the ability of capsaicin to desensitise TRPV1 channels. However, the cream produces a burning sensation immediately following application, presumably due to the acute activation of TRPV1 channels prior to desensitisation.
• Most TRPV1-targeting analgesics are TRPV1 antagonists. The majority of TRPV1 antagonists are developed by modification of capsaicin, and hence are primarily competitive agonists of the capsaicin-binding site. One such drug is capsazepine, which did not see clinical application due to its poor bioavailability and low specificity for TRPV1 channels. Many TRPV1 antagonists followed, showing improved selectivity and more favourable pharmacokinetic properties. A number of TRPV1 antagonists are now in clinical trials.
• Another emerging drug class of TRPV1-targeting drugs are voltage-dependent open-channel TRPV1 blockers such as ruthenium red. They arguably have greater clinical potential than orthosteric antagonists since they are able to selectively target overactive TRPV1 channels. Hence, open channel blockers have a greater potential for blocking TRPV1 action whilst producing minimal adverse effects since they do not block nonpathological TRPV1 activation . However, like capsazepine, ruthenium red shows poor specificity for TRPV1 channels, diminishing the benefit of being an open-channel blocker. There is therefore demand for developing open-channel blockers with greater specificity than ruthenium red.

Question 36

A*:

List 4 subtypes of Na+ channels whose primary function is in pain signalling.

List their specific roles in pain signalling.

What is the potential for drug treatment of these channels?

Answer 36

Subtypes of Na+ channels whose primary function is in pain signalling include Nav1.3, 1.7, 1.8 and 1.9.

• Nav1.7 is primarily expressed in the peripheral branches of DRG neurones, and respond to EPSPs generated by nociceptors at the nerve terminals, amplifying the generator potential. Nav1.7 channels have a relatively hyperpolarised voltage-dependence of activation. Hence, Nav1.7 channels set the pain threshold because the generator potential quickly exceeds the Nav1.7 threshold potential. Like Nav1.3 TTX-resistant channels, and unlike Nav1.8 and 1.9 channels, Nav1.7 channels tend to accumulate in neuromas, contributing to ectopic depolarisation following nerve injury. The role of Nav1.7 channels in pain is evidenced, for example, by the fact that gain-of-function mutations to Nav1.7 channels have been linked to pain disorders such as inherited erythromelalgia and paroxysmal extreme pain disorder (Waxman and Zamponi, 2014). Inherited erythromelalgia is an autosomal dominant condition that is characterised by episodes of burning pain in the extremities. The gain-of-function mutation in inherited erythromelalgia results in a decreased threshold potential, facilitating pain transmission. In paroxysmal extreme pain disorder, a similar gain-of-function mutation results in severe pain in the rectum and face in response to normally nonpainful stimuli. Selective Nav1.7 antagonists have been shown to significantly reduce pain in inherited erythromelalgia in phase 2 clinical trials. Antiepileptics such as carbamazepine have also shown efficacy for the symptomatic treatment of inherited erythromelalgia, however this also results in side effects of nonselective VGSC channel blockade, such as nausea, dizziness and somnolence, that are not present with selective Nav1.7 blockers. On the other hand, expression of Nav1.7 channels is mostly restricted to DRG neurones, making diverse side-effects unlikely, with the most likely contender being anosmia due to the role of Nav1.7 channels in olfaction.
• Like Nav1.7, Nav1.8 is primarily expressed in the peripheral branches of DRG neurones. However, their voltage-dependence of activation is relatively depolarised compared to Nav1.7, (i.e. the threshold potential of Nav1.8 channels is more positive than Nav1.7). Hence, in the rising phase of depolarisation, Nav1.8 channels are responsible for a significant proportion of the total sodium current. There is less evidence for the involvement of Nav1.8 in chronic pain, however gain-of-function mutations are found in small numbers of patients with peripheral neuropathy where Nav1.,7 mutations are absent (Faber et al., 2012). Therefore, Nav1.8 blockade might prove useful in patients that do not respond to Nav1.7 blockade. Numerous Nav1.8 blockers are currently in preclinical stages, and have demonstrated efficacy in rodent models of neuropathic pain.
• The Nav1.9 channel is also expressed in the peripheral branches of DRG neurones, but is also found in the myenteric plexus. Like Nav1.7 channels, Nav1.9 channels have a relatively hyperpolarised voltage-dependence of activation. Furthermore, these channels induce slow inward Na+ currents, and therefore do not contribute significantly to the rising phase of depolarisation. Rather, Nav1.9 channels play a role in extending the pain signal and enhancing excitability. It is believed that Nav1.9 channels play a role in mediating inflammatory pain, since knockout of these channels in mice results in decreased pain response to inflammatory stimuli. No Nav1.9 blockers have yet seen preclinical trials, but may show promise for treating pain in chronic inflammatory conditions such as rheumatoid arthritis, lupus and multiple sclerosis. However, Nav1.9 blockers would be expected to pose a potential risk for GIT side effects due to the involvement of Nav1.9 channels in the enteric nervous system and regulation of GIT function. Hence, if Nav1.9 blockers see clinical trials, the analgesic effect of Nav1.9 blockade will have to be sufficiently large so as to justify foregoing the reduced side effects of Nav1.7 blockade.
• Nav1.3 channels are not expressed in physiological conditions. However, expression of Nav1,3 channels increases in neuromas in response to disturbed peripheral availability of NGF. Nav1.3 has similar kinetics to Nav1.7 in that it has a relatively hyperpolarised voltage-dependence of activation, serving to amplify generator potentials. However, the ability to rapidly repolarise makes Nav1.3 channels able to carry out repetitive firing. Nav1.3 blockers have been in development for a number of years, however very few have demonstrated pharmacokinetic properties that are suitable for clinical use. Development of Nav1.3 blockers might prove useful for the treatment of peripheral nerve injury. Since Nav1.3 expression is limited to pathological conditions, it would not be expected that Nav1.3 blockade would produce any side effects, making them clinically attractive targets.

Question 37

From novel neuronal signalling mechanisms A* cards:

Describe the role of astrocytes in normal pain modulation.

Answer 37

• Astrocytic ATP is converted by ATPases in the extracellular space into adenosine.
• Adenosine binds to A1 receptors in neurones in the ascending pain pathways.
• A1 receptors are Gi/o coupled, and therefore cause inhibition when activated.
• However, there is also evidence of the involvement of other gliotransmitters in pain modulation, as pain threshold was reduced in mice underexpressing glial SNARE proteins (Foley et al.,2011).
• This suggests involvement of other gliotransmitters requiring Ca2+dependent transport, such as glutamate, GABA or D-serine, in potentiating pain transmission.
• This study was met with controversy over the exclusivity of the targeted SNARE protein to astrocytes. However, the targeted SNARE protein was shown to colocalise with the astrocyte-specific protein, GFAP, but not the neuronal-specific protein, NeuN, or microglial-specific protein, Iba1. These are widely used and well-validated markers with high specificity to these cell types, hence the SNARE protein was likely astrocyte-specific.

Question 38

From neuropeptides lecture (neuroceptin, neurostatin and galanin are A*):

Describe the role of neuropeptides such as substance P, CGRP, NAAG, neuroceptin, neurostatin and galanin in the control of pain transmission.

Answer 38

• Substance P and CGRP bind to NK-1 receptors in pain fibres.
• Binding to NK-1 receptors causes PKC-mediated NMDA receptor phosphorylation.
• This increases the affinity of glycine for the glycine site on the NMDA receptor, potentiating NMDA-mediated transmission of pain signals in primary afferent neurones.
• This only occurs when sufficient glutamate is coreleased with the neuropeptide, because sufficient AMPA receptors must first be activated in order to overcome the voltage-dependent Mg2+ block of the NMDA receptors for the neuropeptides to have any effect on transmission.
• NAAG is coreleased with these neuropeptides, which has the ability to bind to presynaptic mGlu3 receptors as a means of negative feedback (NAAG has an analgesic effect).

• Nociceptin and nocistatin are opioid-related neuropeptides that are produced from the same prepropeptide.
• Nocistatin is not biologically active on its own but can antagonise the effects of nociceptin.
• Nociceptin (considered an anti-opioid) causes allodynia, and is therefore a potential target for pain therapy.

• Many neuropeptidergic neurones exhibit strong neuroplasticity.
• In the dorsal root ganglion (DRG), the phenotype of neurones is influenced by peripheral nerve lesions.
• For example, galanin and is upregulated in the DRG in response to nerve injury.
• Galanin has analgesic effects at Gal1 receptors and pronociceptive effects at Gal2 receptors.